Artiodactyl epimorphine

ABSTRACT

An epimorphin protein having action to induce differentiation of milk protein-producing cells into a branched luminal structure in Artiodactyls including cows and sheep and comprising an amino acid sequence set forth in SEQ ID NO: 1 in the Sequence Listing; and a gene encoding said protein are provided. The epimorphin protein of the present invention can be used as a medicament for Artiodactyls or an agent for modifying animal properties. For example, the protein acts to enlarge the mammary gland of cows or sheep to prevent the gland from clogging, thereby a yield of a desired protein secreted in the milk of the animal is increased.

TECHNICAL FIELD

The present invention relates to epimorphin derived from the orderArtiodactyla including pigs, cows, sheep and the like.

BACKGROUND ART

Morphology of various organs of animals is constructed by epithelialtissues, and mesenchymal cells exist around the tissues. An epimorphinis a cell membrane protein that is expressed in mesenchymal cells,particularly in a high amount neighboring epithelial cells (Hirai, Y.,et al., Cell, 69, pp. 471-481, 1992). It is considered that the progressof morphogenesis of the epithelial tissue requires a signal from themesenchymal cells (Gumbiner, B. M., Cell., 69, pp. 385-387, 1992).Epimorphins have been cloned in humans, birds, and rats as well as inmice. The presences of isoforms are known which have a differentsequence of a hydrophobic site (Zha, H., et al., Genomics, 37, pp.386-389, 1996; Hirai, Y., et al., Biochem. Biophys. Res. Commun., 191,p. 1332-1337, 1993; Oka, Y., Developmental Biological Society, 1997,May).

Epimorphin is known to be deeply involved in morphogenesis by epithelialtissues in mice, i.e., differentiation into hair on the fetal talonskin, and differentiation into luminal structure in the fetal lung(Hirai, Y., et al., Cell, 69, pp. 471-481, 1992; Koshida, S., et al.,Biochem. Biphys. Res. Commun., 234, pp. 522-526, 1997). Moreover,epimorphin activates mesenchymal cells and promotes secretion ofcytokines, IL-6 and IL-8 (Oka, Y., et al., Exp. Cell Res., 222, pp.189-198, 1996). Recently, it was revealed that the addition ofepimorphin to milk protein-producing SCp2 cells induces cell growth toform a branched duct structure (Hirai, Y., et al., J. cell Biol., 140,pp. 159-169, 1998). Epimorphin is expected to be effective inelucidating the mechanism of onset of diseases due to abnormality oforgans, developing methods for diagnosing and treating said diseases,generating hairs, lumens, bones, and teeth, generating new bloodvessels, and developing new methods for treatment of injuries (Zha, H.,et al., Genomics, 37, pp. 386-389, 1996; Panaretto, B. A., Reprod.Fertil. Dev., 5, pp. 345-360, 1993; Matsuki, Y., et al, Archs. OralBiol., 40, pp. 161-164, 1995).

DISCLOSURE OF THE INVENTION

A so-called animal factory, which allows an animal to secrete a desiredprotein in its milk, has recently been used practically. Mammals of theorder Artiodactyla including cows and sheep are often utilized asanimals for the secretion of a desired protein in milk. However,particularly when a desired protein has a relatively large molecularweight or when the secretion amount of a desired protein becomes high,mammary glands may often clog and the desired protein cannot beextracorporeally recovered. Accordingly, for practical application of aneffective animal factory, it is required that means be developed whichenlarges the mammary glands of such animals and prevents the mammaryglands of the animals from clogging even when a large amount of thedesired protein is produced in the milk.

The inventors of the present invention have focused on the fact thatepimorphin induces differentiation of SCp2 cells into a branched ductstructure, and conducted various studies to isolate an epimorphin geneof Artiodactyls. As a result, the inventors have succeeded in theisolation of an epimorphin gene derived from an animal belonging toArtiodactyls, and found that the gene product has an activity ofenlarging mammary glands, i.e., an activity of differentiating mammarycells so as to enlarge the internal diameters of the ducts. The presentinvention was achieved on the basis of these findings.

The present invention thus provides a protein comprising an amino acidsequence set forth in SEQ ID NO: 1 in the Sequence Listing (alsoreferred to as “bovine epimorphin 2”), a protein comprising an aminoacid sequence set forth in SEQ ID NO: 3 in the Sequence Listing (alsoreferred to as “bovine epimorphin 4”), and a protein comprising an aminoacid sequence set forth in SEQ ID NO:5 in the Sequence Listing (alsoreferred to as ^(“)sheep epimorphin 2”). The present invention alsoprovides a protein having 95% or more homology to an amino acid sequencefrom the 1st to 262nd amino acids set forth in SEQ ID NO: 1 (bovineepimorphin 2) in the Sequence Listing; a protein having an amino acidsequence from the 1st to 262nd amino acids set forth in SEQ ID NO: 1 inthe Sequence Listing; and a protein having an amino acid sequence fromthe 1st to 262nd amino acids set forth in SEQ ID NO: 5 in the SequenceListing.

The invention further provides a protein having any one of the aminoacid sequences set forth in SEQ ID NOS: 1, 3, and 5 in the SequenceListing wherein one or more amino acids are substituted, deleted, and/oradded, and inducing differentiation of milk protein-producing cellsderived from mammals, preferably those derived from Artiodactyla, into abranched luminal structure; and a protein having any one of the aminoacid sequences of SEQ ID NOS: 1, 3, and 5 in the Sequence Listingwherein one or more amino acids are substituted, deleted, and/or added,and promoting hair growth in mammals, preferably in Artiodactyls.

Furthermore, the present invention provides a protein in which one ormore amino acids are substituted, deleted, and/or added in an amino acidsequence defined by the 1st to 262nd amino acids of either of the aminoacid sequence set forth in SEQ ID NOS: 1 or 5 in the Sequence Listing,which has 95% or more homology to the amino acid sequence defined by the1st to 262nd amino acids of either of the amino acid sequence set forthin SEQ ID NO: 1 or 5 and induces differentiation of milkprotein-producing cells into a branched luminal structure; and a proteinin which one or more amino acids are substituted, deleted, and/or addedin an amino acid sequence defined by the 1st to 262nd amino acids ofeither of the amino acid sequence set forth in SEQ ID NOS: 1 or 5 in theSequence Listing, which has 95% or more homology to the amino acidsequence defined by the 1st to 262nd amino acids of either of the aminoacid sequence set forth in SEQ ID NO: 1 or 5 and promotes hair growth.

From another aspect, the present invention provides a polynucleotideencoding each of the above-mentioned proteins. According to preferredembodiments of the invention, DNAs set forth in SEQ ID NOS: 2, 4, and 6are provided. The invention also provides a DNA comprising continuous 12or more nucleotides contained in said nucleotide sequences in theSequence Listing. The DNA may be either double- or single-strand, andmay be either a sense or antisense strand when the DNA issingle-stranded. Furthermore, the invention provides RNA hybridizing tothe above DNA, and a polynucleotide as being the above polynucleotidewith chemical modification.

The present invention also provides a recombinant vector having theaforementioned polynucleotide, a transformant such as a microbial celland a mammalian cell which comprises said vector, and a process ofproducing the above proteins which comprises the steps of isolating andpurifying the protein from a culture obtained by cultivation of saidtransformant. The present invention further provides an antibody,preferably a monoclonal antibody, recognizing each of the aboveproteins.

BEST MODE FOR CARRYING OUT THE INVENTION

Three isoforms of epimorphins are known to exist (Hirai Y., et al., J.Cell Biol., 140, pp. 159-169, 1998). These isoforms are classified asisoforms 1, 2 and 3, based on the number of amino acids at theC-terminal portion and their properties (hydrophobicity orhydrophilicity). According to this classification, bovine epimorphin 2comprising an amino acid sequence set forth in SEQ ID NO: 1 in theSequence Listing, and sheep epimorphin 2 comprising an amino acidsequence set forth in SEQ ID NO: 5 in the Sequence Listing, which arepreferable examples of the present invention, are both classified asisoform 2. Bovine epimorphin 4 comprising an amino acid sequence setforth in SEQ ID NO: 3 in the Sequence Listing fails to comply with anyone of the above classifications and is recognized as a new type ofisoform.

Structurally, epimorphins can be divided into four domains. Also in thespecification, epimorphins of the present invention are structurallydivided into four portions, each referred to as domain 1, domain 2,domain 3, and domain 4 in the order from the N-terminal side. Domains 1to 4 of the above-mentioned bovine epimorphin 2, bovine epimorphin 4,and sheep epimorphin 2 are as follows (an amino acid sequence set forthin SEQ ID NO:1 in the Sequence Listing will be simply referred to as“Amino acid sequence 1,” which will apply to all the other sequences).

Bovine epimorphin 2

-   Domain 1: 1st to 107th amino acids of amino acid sequence 1-   Domain 2: 108th to 187th amino acids of amino acid sequence 1-   Domain 3: 188th to 262nd amino acids of amino acid sequence 1-   Domain 4: 263rd to 287th amino acids of amino acid sequence 1    Bovine epimorphin 4-   Domain 1: 1st to 107th amino acids of amino acid sequence 3-   Domain 2: 108th to 187th amino acids of amino acid sequence 3-   Domain 3: 188th to 262nd amino acids of amino acid sequence 3-   Domain 4: 263rd to 269th amino acids of amino acid sequence 3    Sheep epimorphin 2-   Domain 1: 1st to 107th amino acids of amino acid sequence 5-   Domain 2: 108th to 187th amino acids of amino acid sequence 5-   Domain 3: 188th to 262nd amino acids of amino acid sequence 5-   Domain 4: 263rd to 287th amino acids of amino acid sequence 5

Domains 1 and 3 are coiled coil regions referred to as “a coiled coilregion on the N side” and “a coiled coil region on the C side”,respectively. Domain 4 is a hydrophobic domain referred to as theC-terminal hydrophobic region. Each of these domains is known to havethe following functions, based on the findings about human and mouseepimorphin.

-   Domain 1: Promotion of differentiation into hair on the fetal talon    skin, differentiation into luminal structure in the fetal lung,    activation of mesenchymal cells, and promotion of secretion of    cytokines, IL-6 and IL-8.-   Domain 2: Cell adhesion and promotion of secretion of GM-CSF (a    growth factor, a type of cytokine-   Domain 3: Function unknown-   Domain 4: Type 1 cell membrane binding domain

Functions of Domains 1 and 2 are engaged in promotion of differentiationof milk protein-producing cells into luminal structure. Functions ofepimorphins and their domains can be determined by methods described inpublications. The present invention further encompasses polypeptidescorresponding to each of the domains explained as for bovine epimorphin2, bovine epimorphin 4, and sheep epimorphin 2; and a polypeptide havingone of the amino acid sequences of each of these domains, in which oneor more amino acids are substituted, deleted, and/or added, and having abiological action substantially the same as that of each of the domains.

As for the above three types of epimorphin, the amino acid sequence fromthe 1st to 262nd amino acids of the bovine epimorphin 2 is completelyidentical to that from the 1st to 262nd amino acids of bovine epimorphin4. Furthermore, this sequence has 99.2% homology to the sequence fromthe 1st to 262nd amino acids of sheep epimorphin 2 and the sequences arewell conserved. Therefore, the region of the amino acid sequences is acharacteristic amino acid sequence for Artiodactyla epimorphin, and aprotein comprising said amino acid sequence is a preferred embodiment ofthe present invention. Any proteins having homology of 95% or more,preferably 98% or more, with the amino acid sequence from the 1st to262nd amino acids of the bovine epimorphin 2, and having a functionsubstantially equivalent to that of the above amino acid sequence fallwithin the scope of the present invention. In addition, sheep epimorphin2 and human epimorphin have 94.3% homology in an amino acid sequencefrom the 1st to 262nd amino acids, which are also well conserved. Theterm “homology” herein used means the maximum value obtained bysubjecting one amino acid sequence to alignment based on the othersequence where the two sequences are compared. Such alignment can beconducted conveniently by using a commercially available computersoftware. An example of the software includes Genetics-Mac sold bySoftware Developing Co., Ltd.

The present invention also encompasses within the scope a protein havingany one of the amino acid sequences set forth in SEQ ID NOS: 1, 3, and 5in the Sequence Listing, in which one or more amino acids aresubstituted, deleted and/or added, and inducing differentiation of milkprotein-producing cells into branched luminal structure; and a proteinhaving any one of the amino acid sequences set forth in SEQ ID NOS: 1,3, and 5 in the Sequence Listing, in which one or more amino acids aresubstituted, deleted and/or added, and promoting hair growth (theseproteins are herein also referred to as “modified proteins” and where“protein(s) of the present invention” is referred to, the description isused to also encompass these modified proteins unless otherwisespecifically mentioned). The action of inducing differentiation of milkprotein-producing cells into branched luminal structure can bedetermined by methods described in J. Cell Biol., 140, pp. 159-169,1998. It is also recognized that known epimorphins have hair growthpromoting action (Hirai, Y., et al., Cell, 69, pp. 471-481, 1992).

These modified proteins can be produced by treating Escherichia colihaving a DNA encoding any one of the amino acid sequences set forth inSEQ ID NOS: 1, 3 and 5 with an agent such asN-nitro-N-nitro-N-nitrosoguanidine to cause mutation, recovering a geneencoding a modified protein from the bacterial cells; and performing anordinary gene expression. In addition, the gene may be directly treatedwith an agent such as sodium sulfate, or nucleotide deletions,substitutions or additions may be directly introduced into the gene bytechniques such as site-specific mutation method (Kramer, W. et al.,Methods in Enzymology, 154, 350, 1987) and recombinant PCR method (PCRTechnology, Stockton press, 1989).

Methods to obtain genes encoding the protein of the present inventionare not particularly limited. For example, a genetic DNA can beefficiently recovered by methods specifically described in the examplesof the specification. It is well known to persons skilled in the artthat, due to degeneracy of genetic codes, at least a part of nucleotidesof the nucleotide sequence of the polynucleotide can be replaced withother types of nucleotides without changing the amino acid sequence ofthe polypeptide produced based on the polynucleotide. Therefore, thepolynucleotides of the present invention encompass any epimorphin genesencoding any one of the amino acid sequences set forth in SEQ ID NOS: 1,3, and 5. As preferable examples of the genes of the present invention,DNAs encoding bovine epimorphin 2, bovine epimorphin 4, and sheepepimorphin 2 are shown in SEQ ID NOS: 1, 4, and 6, respectively.

Antisense polynucleotides and derivatives thereof also fall within thescope of the present invention, which have a nucleotide sequence of anantisense strand of the polynucleotide encoding the protein of thepresent invention. Although the antisense polynucleotides are providedas a class of an embodiment of the aforementioned polynucleotides, thepolynucleotides may also be herein referred to as “antisensepolynucleotides” to specify that they are polynucleotides particularlyhaving a nucleotide sequence of an antisense strand. The antisensepolynucleotide can hybridize to a polynucleotide encoding each of theabove proteins, and where a polynucleotide to which the antisensepolynucleotide hybridizes is a polynucleotide in a coding region,biosynthesis of a polypeptide encoded by said polynucleotide can beinhibited.

An antisense polynucleotide for inhibiting the biosynthesis of thepolypeptide preferably comprises 12 or more nucleotides, furtherpreferably comprises 16 or more nucleotides. An unnecessarily longsequence is not preferred to achieve incorporation of the full length ofan antisense polynucleotide into cells. For intracellular incorporationof the antisense polynucleotide to induce inhibition of the biosynthesisof the above proteins, the antisense polynucleotides comprising 12 to 30nucleotides, preferably 15 to 25 nucleotides, more preferably 18 to 22nucleotides may preferably be used.

The antisense polynucleotides or derivatives thereof of the presentinvention encompass any of those comprising multiply bound nucleotideseach consisting of a base, a phosphoric acid, and a sugar, regardless ofthey are naturally or non-naturally derived. Typical examples include anatural antisense DNA and antisense RNA. Examples of non-naturalpolynucleotides include methylphosphonate-type and phosphorothioate typepolynucleotides. Various antisense polynucleotide derivatives havingexcellent abilities of binding to a target DNA or mRNA, tissuespecificity, cell-permeability, nuclease resistance, and intracellularstability can be obtained by applying methods in antisense technologyavailable to persons skilled in the art.

Generally, from a viewpoint of easy hybridization, it is preferable todesign antisense polynucleotides and derivatives thereof having anucleotide sequence complementary to that in a region forming an RNAloop. In addition, an antisense polynucleotide that has a sequencecomplementary to a sequence of around a translation initiating codon, aribosome binding site, a capping site, or a splicing site can beexpected to have a high suppressing effect on expression. Among theantisense polynucleotides and derivatives thereof, those comprising asequence complementary to genes encoding each of the above proteins, orto a sequence of around a translation initiation codon by mRNA, aribosome binding site, a capping site, and/or a splicing site arepreferred from a viewpoint of the effect on the suppression ofexpression.

Among polynucleotide derivatives widely known to date, an example of thederivatives having at least one enhanced abilities of nucleaseresistance, tissue specificity, cell-permeability, and binding abilityincludes a derivative having phosphorothioate bondings as a backbonestructure. The polynucleotides and derivatives thereof of the presentinvention encompass the aforementioned derivatives having thesefunctions or said structure.

Among the antisense polynucleotides of the present invention, naturalantisense polynucleotides can be prepared by using a chemicalsynthesizer, or by carrying out the PCR using a DNA encoding each of theabove proteins as a template. The methyl phosphonate-type orphoshorothioate-type polynucleotide derivatives can generally beproduced by chemical synthesis. For the synthesis, operations may beconducted according to an instruction manual appended to a chemicalsynthesizer, and a resulting product synthesized can be purified by aHPLC method using reverse phase chromatography and the like.

The polynucleotide encoding the protein of the present invention, andthe antisense polynucleotides or portions thereof (a polynucleotidehaving a nucleotide sequence which comprises continuous 12 or morenucleotides) can be used as probes for screening the gene of the presentinvention from a cDNA library or the like. For said purpose, apolynucleotide with a GC content of 30 to 70% can preferably be used. Apolynucleotide having a sequence comprising continuous 16 or morenucleotides may particularly preferred. Derivatives of thepolynucleotides may be used as probes. In general, a sequence havingnucleotides of the aforementioned number of nucleotides is recognized asa sequence with specificity.

As the cDNA libraries used for the screening with the above probe, thoseconstructed from mRNA may preferably used. A class of cDNAs selected byrandom sampling from the above cDNA libraries may be used as a samplefor the screening. Commercially available libraries may also be used.For example, DNA having a nucleotide sequence comprising a continuous 12or more nucleotides of the nucleotide sequence set forth in SEQ ID NO:2, 4 or 6 in the Sequence Listing, or a polynucleotide (antisensepolynucleotide) that hybridizes to the DNA can be used as a probe forscreening a DNA encoding any one of the amino acid sequences set forthin SEQ ID NOS: 1, 3 and 5 from the cDNA library or the like.

Moreover, a tissue in which an mRNA deriving from the gene of thepresent invention is expressed can be detected by means of Northernblotting hybridization for mRNAs deriving from various tissues using apolynucleotide encoding the protein of the present invention or anantisense polynucleotides thereof, or a fragment polynucleotide thereofas a probe.

Transformants can be prepared by inserting a cDNA capable of hybridizingto the gene of the present invention into an appropriate vector, andthen introducing the recombinant vector into a host (for example,Escherichia coli). Types of the vectors and the hosts are notparticularly limited, and an appropriate expression vector can beselected depending on the type of the host. As the host, any of bacteriasuch as Escherichia coli, yeast, or animal cells can be used. Animalcells are preferably used, and most preferably, mammal cells may beused. Methods for introducing the recombinant vector into an appropriatehost such as Escherichia coli to obtain a transformant are notparticularly limited. Any technique available to those skilled in theart can be employed.

The protein of the present invention can be produced by culturing thetransformant, in which the gene of the present invention is introduced,to allow the cells to amplify the gene DNA or express the protein. Avariety of literature and reports are available about the production andculture of transformants, and various techniques have been developed andwidely used in the art. Accordingly, those skilled in the art can easilyproduce the protein of the present invention based on the nucleotidesequences set forth in the specification. As methods for introducinggenes into cells, calcium chloride method, protoplast method,electroporation and the like may be used. Nuclear microinjection may bemost preferably used.

Separation and purification of the target protein from a culture can beperformed by the appropriate combination of techniques available topersons skilled in the art. For example, by performing proceduresincluding concentration, solubilization, dialysis, and various types ofchromatographies as required, the protein of the present invention canbe efficiently recovered and purified. More specifically, the separationand purification may be carried out by appropriately choosingimmunoprecipitation, salting out, ultrafiltration, isoelectricprecipitation, gel filtration, electrophoresis, ion exchangechromatography, various affinity chromatography such as hydrophobicchromatography or antibody chromatography, chromatofocusing, adsorptionchromatography, and reverse phase chromatography.

The protein of the present invention can be produced as a fusion peptidewith other polypeptide(s). It should be understood that such fusionpolypeptides also fall within the scope of the present invention. Typesof the polypeptide to be fused are not particularly limited. An exampleincludes a signal peptide which promotes extracellular secretion. Thepreparation of the fusion protein can also be preformed by usingtransformants. When the protein or the modified protein of the presentinvention is produced by using the fusion protein, the fusion protein istreated with a chemical substance such as cyanogen bromide or an enzymesuch as a protease, and then the cleaved target product may be separatedand purified.

A fusion protein may also be prepared which composes of a partialpolypeptide responsible for epimorphin-like biological activities in theprotein or modified protein of the present invention (so called anactive domain) and other polypeptide. The above-mentioned domain 1and/or domain 2 can be used as such active domains. For example, asoluble polypeptide containing an active domain can be produced byremoving domain 4, and a fusion protein composed of the solubilizedactive domain and other polypeptide (e.g., a signal peptide) can beproduced. A chimeric epimorphin may be produced by appropriatelycombining plural domains selected from each of the above domains of theprotein of the present invention and each of the domains of the othertypes of epimorphin. Furthermore, a fusion protein may be produced bybinding other polypeptide to a polypeptide composed of an appropriatecombination of each of the above domains.

An antibody recognizing the protein of the present invention can beproduced by using the protein of the present invention or a partialpolypeptide chain thereof. The antibody of the present invention can beproduced by sensitizing and immunizing a mammal with the protein of thepresent invention according to methods widely used in the art. Whetheror not the antibody can recognize the protein of the present inventionis determined by a method such as Western blotting, ELISA, orimmunostaining (e.g., measurement by FACS). As immunogens, the proteinof the present invention as well as a part of the protein bound to acarrier protein, e.g., bovine serum albumin may be used. The part of theprotein of the present invention may preferably comprise eight or moreamino acid residues, and such polypeptide may be synthesized, forexample, by using a peptide synthesizer.

A monoclonal antibody generated by a hybridoma which is produced byusing lymphocytes of an immunized animal may be used as the antibody ofthe present invention. Methods for preparing monoclonal antibodies arewell known in the art and widely used (Antibodies A Laboratory Manual, aCold Spring Harbor Laboratory Press, 1988, Chapter 6). In addition,active fragments of the above antibody can be used as the antibody ofthe present invention. In the specification, “active fragments” meansfragments of an antibody having antigen-antibody reaction activity. Morespecifically, examples include F(ab′)₂, Fab′, Fab, and Fv. For example,where the antibody of the present invention is decomposed with pepsin,F(ab′)₂ is obtained; where decomposed with papain, Fab is obtained.Where F(ab′)₂ is reduced with a reagent such as 2-mercaptoethanol andthen alkylated with monoiodoacetic acid, Fab′ is obtained. Fv is amonovalent active antibody fragment composed of a heavy-chain variableregion and a light-chain variable region bound with a linker to eachother. A chimeric antibody can be obtained by preserving these activefragments and replacing the other part with fragments deriving fromother animals. Any of the above described antibodies and activefragments and the like fall within the scope of the present invention.

The protein of the present invention can be detected by a methodutilizing an antibody or an enzymatic reaction. Examples of the methodsfor detecting the protein of the present invention using an antibodyinclude (I) a method for detecting the protein of the present inventionusing the aforementioned antibody with labeling, and (II) a method fordetecting the protein of the present invention using the aforementionedantibody and a labeled secondary antibody recognizing said antibody. Asmeans for labeling, radioactive isotopes (R1), enzymes, avidin orbiotin, or fluorescent materials (FITC, rhodamine, and the like) may beutilized. Examples of methods utilizing an enzymatic reaction includeELISA, immunoagglutination, Western blotting, a process for identifyingan immunoreactive molecule using flow cytometry and methods similarthereto.

EXAMPLES

The present invention will be explained more specifically by way ofexamples. However, the scope of the present invention is not limited tothe following examples.

The sheep and bovine epimorphin genes of the present invention wereobtained by the methods set out below.

1. Isolation of epimorphin cDNA

1) DNA to be used as a probe was labeled using Random Primed DNALabeling Kit (manufactured by Boehringer Manheim) according to theinstructions appended to the kit. DNA comprising the full length of anucleotide sequence of a mouse epimorphin-coding region was used as aDNA probe.

2) DNA reaction solution having the following composition was thenprepared. The solution was incubated for 30 minutes at 37° C., and thenheated for 10 minutes at 65° C. for inactivation of enzymes.

DNA probe (50 ng/μl)  2 μl H₂O  7 μl dNTPs  3 μl [α-³²P]d-CTP(370MBq/ml) (manufactured by  5 μl Amersham Pharmacia Biotech) Primer  2μl Klenow enzyme  1 μl Total 20 μl

3) The reaction solution was centrifuged with a Centri-Sep spin column(manufactured by Princeton Separations, Inc.) swollen with H₂O, therebyobtaining a labeled DNA probe.

4) Using the sheep lung and cow lung as libraries (Uni-ZAPXR Library,ST, manufactured by STRATAGENE), phage plaques were obtained by astandard method (New Cell Technology Experimental Protocol, Shu-junsha).

5) The labeled DNA probe obtained in 3) were measured by using ascintilation counter, added to a hybridization reaction solution to1×10⁶ cpm/ml. Then, the product was allowed to react with a nylonmembrane to which cDNA derived from the plaque was immobilized(Hybond-N+, manufactured by Amersham Pharmacia Biotech).

Prehybridization

-   -   Reaction solution: ExpressHyb (manufactured by Clontech        Laboratoreis, Inc.)    -   Reaction temperature: 68° C.    -   Reaction time: 30 minutes        Hybridization    -   Reaction solution: ExpressHyb (manufactured by Clontech        Laboratories, Inc.)    -   ³²p labeled cDNA probe: 1×10⁶ cpm/ml    -   Reaction temperature: 68° C.    -   Reaction time: 1 hour

6) The membrane after the hybridization was washed with the solution asdescribed below according to the protocols for ExpressHyb (manufacturedby Clontech Laboratories, Inc.)

-   -   2×SSC, 0.05% SDS 500 ml    -   Room temperature    -   Time: 40 minutes    -   0.1×SSC, 0.1% SDS    -   50° C.    -   Time: 40 minutes

7) The washed nylon membrane was exposed to X-ray film (e.g., XAR 5 filmmanufactured by Eastman Kodak Company) overnight at −80° C. to takeautoradiographs.

8) Positions of positive plaques were determined based on the resultingautoradiographs, and then phages in the corresponding plaques on theagar were recovered in an SM solution.

9) The recovered phages were again allowed to form plaques on a NZY agarmedium by a standard method, and then immobilized on a nylon membrane.

10) Procedures 5) to 9) were repeated 3 times to obtain unified phagesin positive plaques. The phages were collected and suspended in 500 μlof SM solution. The suspension was added with 20 μl of chloroform andstirred to prepare a phage solution. cDNA isolated from the phagesolution contained sheep and bovine epimorphin genes.

2. Preparation of sheep and bovine epimorphin cDNA in large quantity

1) 10 μl of the phage solution suspended in the SM solution, 200 μl ofXL1-Blue Escherichia coli (manufactured by STRATAGENE), and 1 μl ofhelper phages (manufactured by STRATAGENE) were mixed and allowed toreact for 15 minutes at 37° C.

2) The mixed solution was then transferred in 3 ml of LB medium, and themedium was shake-cultured overnight at 37° C. to cleave and recover thegenes as pBluescript phagemid.

3) The phagemid was treated for 20 minutes at 70° C. and centrifuged at1000 rpm for 15 minutes, and then the supernatant was recovered.

4) 100 μl of the supernatant and 200 μl of SOLR Eseherichia coli weremixed, and then the mixture was allowed to react for 15 minutes at 37°C.

5) 10 μl of the solution obtained in 4) was inoculated over a plate ofLB medium containing 50 μg/ml of ampicillin, and then the plate wascultured overnight at 37° C.

6) One colony was added to 3 ml of an LB medium containing 50 μg/ml ofampicillin, and then the medium was shake-cultured overnight at 37° C.

7) The culture product was centrifuged at 2000 rpm for 10 minutes torecover the E. coli.

8) The E. coli was purified using a Plasmid Mini Kit (manufactured byQIAGEN) to prepare plasmid DNA containing sheep or bovine epimorphingenes in a large quantity.

3. Sequencing of sheep and bovine epimorphin cDNA The entire nucleotidesequences of sheep and bovine epimorphin genes in the plasmid DNA weredetermined by the dye terminator method using an auto sequencer.

4. Determination of amino acid sequences

Amino acid sequences of sheep and bovine epimorphin were determinedbased on the nucleotide sequences determined in the above step 3(Genetics-Mac provided by Software Development Co., Ltd. was used as acomputer software for this step).

Each of the amino acid sequences was named; sheep epimorphin 2, bovineepimorphin 2, and bovine epimorphin 4.

5. Preparation of transformants

cDNA of each of sheep epimorphin 2, bovine epimorphin 2, and bovineepimorphin 4 was inserted into pBluescript SK(−) plasmid, and then theplasmid was introduced in Escherichia coli strain SOLR to obtaintransformants. The resulting respective transformants were named asSh-EPM2, Bo-EPM2, and Bo-EPM4.

INDUSTRIAL APPLICABILITY

The protein of the present invention can exert epimorphin-likebiological nactivities including, for example, differentiation of milkprotein-producing cells into branched luminal structure, and promotionof hair growth. The protein of the present invention can be used as amedicament for Artiodactyls or as an agent for modifying animalproperties. For example, the protein of the present invention enlargesthe mammary glands of Artiodactyls including cows and sheep to preventthe gland from clogging, thereby an yield of a desired protein secretedin the milk of the animal is increased. Furthermore, sheep for a highproductivity of wool and transgenic animals (an animal factory) for ahigh productivity of a target protein can be generated by using the geneof the present invention.

1. An isolated protein comprising any one of the amino acid sequencesset forth in SEQ ID NOS: 1, 3, and 5 in the Sequence Listing.
 2. Anisolated protein having 98% or more homology to an amino acid sequencefrom the 1st to 262nd amino acids of the amino acid sequence set forthin SEQ ID NO: 1 in the Sequence Listing, and which has an activity ofenlarging mammary glands.
 3. The protein according to claim 2 having anamino acid sequence from the 1st to 262nd amino acids of the amino acidsequence set forth in SEQ ID NO: 1 in the Sequence Listing.
 4. Theprotein according to claim 2 having an amino acid sequence from the 1stto 262nd amino acids of the amino acid sequence set forth in SEQ ID NO:5 in the Sequence Listing.
 5. An isolated protein having any one of theamino acid sequences set forth in SEQ ID NOS: 1, 3 and 5 in the SequenceListing wherein one or more amino acids is substituted, which has 98% ormore homology to the amino acid sequence disclosed in SEQ ID NOS: 1, 3or 5 and which induces differentiation of a milk protein-producing cellinto a branched luminal structure.
 6. An isolated protein in which oneor more amino acids is substituted in an amino acid sequence defined bythe 1st to 262nd amino acids of either of the amino acid sequence setforth in SEQ ID NOS: 1 or 5 in the Sequence Listing, which has 98% ormore homology to the amino acid sequence defined by the 1st to 262ndamino acids of either of the amino acid sequence set forth in SEQ ID NO:1 or 5 and induces differentiation of a milk protein-producing cell intoa branched luminal structure.
 7. An isolated protein having any one ofthe amino acid sequences set forth in SEQ ID NOS: 1, 3, and 5 in theSequence Listing wherein one or more amino acids is substituted whichhas 98% or more homology to the amino acid sequence disclosed in SEQ IDNOS: 1, 3 or 5 and which promotes hair growth.
 8. An isolated protein inwhich one or more amino acids is substituted in an amino acid sequencedefined by the 1st to 262nd amino acids of either of the amino acidsequences set forth in SEQ ID NOS: 1 or 5 in the Sequence Listing, whichhas 98% or more homology to the amino acid sequence defined by the 1stto 262nd amino acids of either of the amino acid sequences set forth inSEQ ID NO: 1 or 5 and promotes hair growth.
 9. An isolatedpolynucleotide encoding the protein according to claim
 1. 10. Anisolated polynucleotide according to claim 9, which is a DNA set forthin SEQ ID NO: 2, 4 or
 6. 11. An isolated protein having 98% or morehomology to an amino acid sequence form the 1st to 262nd amino acids ofthe amino acid sequence set forth in SEQ ID NO:1in the Sequence Listing,and which promotes hair growth.